About the Project
Epithelial tissues are essential structures in animals, creating compartments and barriers such as the skin and lining of the gut. Often such protective tissues cease mitosis and switch to endoreplication, leading to highly polyploid nuclei. Hypotheses on the relevance of polyploidy for tissue function include: (a) larger nuclei support larger cell sizes that help maintain barrier tissue integrity, (b) simultaneous transcription from multiple gene copies can support rapid synthesis of important proteins. On the other hand, endoreplication can lead to a terminally differentiated cell state that impairs competence for apoptosis in aged cells, posing a challenge for the efficient, controlled demise of specific tissues at the end of the tissue’s lifetime. This project uses developmental genetic approaches to test the relevance of the two hypotheses on the functional value of polyploidy as well as the retention of apoptosis competence in an excellent experimental model: the extraembryonic epithelia of insect embryos.
Insect eggs are laid externally in a range of environments, and their extraembryonic (EE) tissues protect and support the developing embryos. There are two EE tissues, the outer serosa and the inner amnion, where the latter forms a fluid-filled cavity around the embryo in an analogous way to its namesake in amniote vertebrates (including humans). The serosa and amnion are each polyploid to characteristic levels, with especially large nuclei in the serosa. Given the protective and physiological requirements of the EE tissues, both large cell size and rapid transcription could be relevant to their function. Genetic alterations to cell number and geometry in each offer a powerful experimental approach to examine the link between ploidy level and barrier integrity: do impaired and physically challenged tissues increase their ploidy to regenerate an intact barrier? Equally, both of these tissues undergo a spatially and temporally precise progression of apoptosis that is highly regulated and tightly coordinated with the requirements of the maturing embryo. How is this possible at the end of these tissues’ lifetimes, in aged polyploid cells?
This project will use a range of techniques, including RNA interference (RNAi) and live cell imaging, to functionally test key genes’ roles in regulating EE tissue polyploidy. The primary research organism for this work is the red flour beetle, Tribolium castaneum, where we have established an excellent genetic toolkit, including transgenic GFP labelling of each EE tissue and visualization tools to detect active apoptosis. This supports detailed, quantitative analyses of cell and tissue structure from multi-dimensional bioimaging data, to evaluate intact barrier epithelia and those with compromised integrity. Furthermore, our recent RNA-seq analyses and pilot functional testing have identified a number of candidate genes to examine in testing hypotheses on tissue-specific polyploidy. Complementing the in vivo imaging work, ploidy dynamics will also be directly measured with techniques such as RT-qPCR, flow cytometry (FACS), and next generation sequencing methods.
BBSRC Strategic Research Priority: Integrated Understanding of Health: Ageing
Techniques that will be undertaken during the project:
Multi-dimensional live cell imaging microscopy
RNA interference (RNAi)
(Fluorescent) histology for gene expression and cellular structure
Computational work for image processing and quantitative analysis
Orr-Weaver, T.L. (2015). When bigger is better: the role of polyploidy in organogenesis. Trends Genet. 31, 307-315.
Hilbrant, M., Horn, T., Koelzer, S., and Panfilio, K.A. (2016). The beetle amnion and serosa functionally interact as apposed epithelia. eLife 5, e13834.
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